Project Summary/Abstract (Modifications to text are denoted in italics) Obesity is a worldwide epidemic affecting 600 million people and is associated with a cluster of metabolic diseases such as diabetes and insulin resistance. Hence, obesity and associated disorders are increasingly becoming a societal burden that requires a deeper understanding of the physiology and the development of robust new treatments. One of the culprits in metabolic disease pathogenesis is dysregulation of lipid metabolism and signaling at the adipose tissue and the whole organism level. In a lipidomics analysis on adipose tissue GLUT4 overexpressing (AG4OX) mice, that are resistant to developing metabolic disease, a novel class of bioactive lipids called fatty acid hydroxy fatty acids (FAHFAs) were identified. Circulating levels of a sub-class, palmitic acid hydroxy fatty acids (PAHSAs) are negatively correlated with insulin resistance in rodents and humans suggesting a new aspect in lipid dysregulation in metabolic dysfunction. Furthermore, oral administration of PAHSAs improve glucose tolerance in diet-induced obese mice by increasing glucagon-like peptide 1 (GLP-1) and insulin secretion, inhibiting adipose tissue inflammation, and improving insulin sensitivity. Discovery of FAHFAs and their function in metabolic responses led to interest in their therapeutic potential, and regulation and activity. This project will integrate biochemical, cell culture and mouse model systems to understand the regulation and activity of FAHFAs in the alleviation of metabolic disease. Recently, a new FAHFA hydrolase, androgen-induced gene 1 (AIG-1), was identified that preferentially hydrolyzes FAHFAs in vitro suggesting AIG1 can regulate FAHFA levels and metabolic function in vivo. In aim 1, the role of AIG-1 in FAHFA regulation and metabolism will be evaluated. For this purpose, FAHFA levels will be measured in WT and AIG1-/- mice to test whether their levels are increased in AIG1 deficiency. Next, WT and AIG1-/- littermates will be characterized for their imetabolic phenotype in order to test the hypothesis that endogenous upregulation of FAHFAs improves metabolic dysfunction. Preliminary data demonstrated that PAHSAs exert their beneficial effects at least partially via the lipid receptor, GPR120 leading to insulin-dependent glucose uptake in adipocytes. It is yet to be determined whether FAHFAs are endogenous GPR120 ligands and whether their activity is dependent on GPR120 signaling. In aim 2, the role of GPR120 signaling in FAHFA action will be evaluated. GPR120-/- cell and mouse models will be utilized to stratify the contribution of GPR120 activity to metabolic benefits exerted by FAHFAs by administration of FAHFAs to WT and GPR120-/- experimental groups and analysis of metabolic as well as inflammatory phenotype. Overall, these studies will expand the knowledge on the regulation of these remarkable lipids and dissect mechanisms by which they regulate insulin sensitivity and glucose metabolism. Results of this study have the potential to identify new targets related to FAHFA modulation for prevention and treatment of metabolic diseases associated with obesity.